Method of forming multiwell filtration plates

ABSTRACT

A method of forming a multiwell filtration plate comprising of first gluing a filter to a major surface of a multiwell plate so as to close off one entrance to the wells of the plate. The filter is then heat sealed so as to collapse the pores of the filter in the area between the wells so as to prevent lateral migration of fluid from one well to another.

The present invention relates to a method of forming a multiwellfiltration plate. More particularly, it relates to a method of forming amultiwell filtration plate using glue and heat to bond the filter to theplate.

BACKGROUND OF THE INVENTION

Multiwell plates have existed for many years. Most are in multiples of6, 8 or 12 such as 24, 96 or 384 wells in a single plate.

Methods to attach the filter material to the bottom of the plate so asto seal off the bottom of each well by the porous filter material haveincluded mechanical means such as friction fitting of individual piecesin each well or clips or edge bands to hold the filter material againstthe bottom; adhesives, heat bonding, over molding and thermal bondingvia an under drain.

Each approach has its drawbacks. Simply stuffing a cut piece into eachwell is time consuming and provides less than 100% sealing accuracy.With the 384 well format, this approach is impractical. Likewise, usinga clamp or edge band allows the filter material in the middle toseparate from the bottom of the wells allowing for cross talk orcontamination between wells.

Adhesives require proper placement and alignment of the adhesive andfilter material so as to prevent adhesives from spreading into the areaof filter inside the well that reduces its active filtration area.Moreover, adhesives do not extend through the entire thickness of thefilter material allowing for cross talk and contamination between wellsthrough the filter material beyond the glue.

Overmolding or insert molding eliminates cross talk and forms anintegral well, but it is costly to set up and run and is a relativelyslow process. Moreover, its use at smaller well sizes (384+) is limitedby the ability to form channels and gates for the introduction of themolten plastic in that small area.

Using thermal energy to bond and seal the filter to the bottom of theplate is difficult. Achieving 100% sealing of the filter to the bottomby thermal bonding is not possible. Some filter materials do not bondproperly to the material of the plate, limiting this approach to onlycompatible materials. Other filters are extremely heat sensitive makingthis approach untenable as the filter structure tends to collapse tosuch an extent that active filtration area is compromised.

Using thermal energy to trap the filter between an upper plate and lowerplate is possible. Again, it is limited in speed and cost to set up andrun. Moreover, it requires the use of a fixed design for a bottom platethat may either be unnecessary or improperly suited for the desiredapplication.

What is desired is a process that is fast, inexpensive and reliable formaking a multiwell filtration plate where each well is integrally sealedabout its edge and no cross talk or contamination between wells ispossible. The present invention provides such a process.

SUMMARY OF THE INVENTION

The present invention comprises a process for forming integral sealedwells in a multiwell plate by first gluing a filter material to thebottom of a multiwell plate containing a series of two or more wellsopen at the top and bottom of the plate and after gluing the filter inplace sealing and bonding the filter material by a heat sealing processalong the bottom surface of the plate so as to collapse the porousstructure of the filter in the areas outside of the wells. Optionally, adirector plate may then be glued to the filter side of the plate alongthe collapsed regions. The process provides a multiwelled device havinga filter attached to its bottom surface wherein each well is integraland fully sealed so that no cross talk or contamination occurs betweenadjacent wells.

IN THE DRAWINGS

FIG. 1 shows a first embodiment of the invention in cross-sectionalview.

FIG. 2 shows a close up cross-sectional view of the embodiment of FIG. 1taken along lines 1—1.

FIG. 3A shows a planar view of a bottom surface of the present inventionas described in Example 2.

FIG. 3B shows a planar view of a bottom surface of a comparative plateas described in Example 2.

FIG. 4A shows a planar view of a bottom surface of the present inventionas described in Example 3.

FIG. 4B shows a planar view of a bottom surface of a comparative plateas described in Example 3.

FIG. 5 shows a top down planar view of a 345 well, 96 active well platedesign mentioned in Example 3.

DETAILED DESCRIPTION

The present invention is a process for forming a multiwell plate havinga filter attached to its bottom surface in a manner that provides anintegral seal around the outer periphery of each well.

FIG. 1 shows a partial cross sectional view of a first embodiment of thepresent invention. As shown, plate 2 has a top surface 4, a bottomsurface 6 and a thickness 8 between. A series of wells 10 are formedthrough the thickness 8 of the plate 2. Each well 10 has an upperopening 12 corresponding with the upper surface 4 of the plate and alower opening 14 corresponding with the lower surface 6 of the plate andthe well 10 forming a through hole through the thickness 8 of the plate2 from the top opening 12 to the bottom opening 14.

A filter sheet 16 is attached to the bottom surface 6 of the plate 2such that it covers all of the bottom openings 14 of the wells 10 andthe surrounding bottom surface 6 of the plate. The area of the filter16, which is in contact with the bottom surface 6, has been sealed andadhered to the bottom surface 6 by a sealed area 18.

FIG. 2 shows a close up cross sectional view of the embodiment ofFIG. 1. The plate 2 again has a top surface 4, a bottom surface 6, athickness 8, series of wells 10 and the membrane 16 attached to thebottom surface 6 as described in relationship to FIG. 1. As shown inFIG. 2, the sealing area 18 is formed of two aspects; a glue attachment20 between the bottom surface 6 of the plate 2 and at least a portion ofthe thickness of the filter 16 and a heat seal 22 formed between theouter surface 24 of the filter 16 and the glue attachment 20.

The heat seal, at the very least, creates a liquid impermeable barrieraround each of the wells. Preferably, it causes the porous structure ofthe filter outside of the wells to substantially collapse reducing theporosity in those areas to substantially nothing. This coupled with theglue attachment effectively forms an impermeable dam around the outerperiphery of each well so that the liquid in a well does not movelaterally to an adjacent well, thus eliminating cross talk and thepotential for contamination.

The method for forming a plate of FIG. 1 is as follows: One takes aplate having a top surface, a bottom surface and defined thicknessbetween the two surfaces. The plate has a series of wells runningthrough the thickness of the plate and having a top and bottom openingcorresponding with the top and bottom surface of the plate.

An adhesive is applied to the bottom surface of the plate (e.g. to thesolid portions of the plate bottom) and the filter is then placed on topof the adhesive and compressed to make intimate contact with theadhesive. Preferably, the adhesive penetrates a portion of the thicknessof the filter material to establish a good bond between the plate bottomand the filter. Alternatively, one could apply the glue via a roboticX/Y applicator to the filter and then attach the filter to the plate.However in this embodiment, one must carefully align the filter with theplate to ensure good sealing. The use of alignment pins, pins/holes,notches, marks and the like are useful if one decides to use thisalternative embodiment.

After curing, the filter is then subjected to a heat sealing step. Thearea of the filter that is directly over a solid portion of the platebottom is heat sealed. The area of the membrane that is over the openwells in the plate bottom remain substantially free of either theadhesive or the heat bonding so as to retain the maximum area offiltration for each well.

If desired, an under drain containing collector wells or directingspouts that are in register with the wells of the plate may then beattached to the bottom of the plate using an adhesive, heat weld,vibration weld and the like.

The plate may be any plate commonly used in multi well filtration. Itshould contain a series of two or more wells. Preferably, it contains aseries of wells that are at least 12, preferably 24, more preferably atleast 96, or at least 384 and even up to 1,536 wells per plate. Theplate should be relatively rigid or self-supporting to allow for easyhandling during manufacturing and easy handling during use by the enduser (a human or a robot). Additionally, its dimensions should conformto those set out by the Society for Biological Standards so that it canbe used in all robotic applications. Preferably the plate may be made ofpolymeric, especially thermoplastic materials, glass, metallicmaterials, ceramic materials, elastomeric materials, coated cellulosicmaterials and combinations thereof such as epoxy impregnated glass mats.In a more preferable embodiment, the plate is formed of a polymericmaterial including but not limited to polyethylene, acrylic,polycarbonate and styrene. The wells can be made by injection molding,drilling, punching and any other method well known for forming holes inthe material of selection. Such plates are well known and commerciallyavailable from a variety of sources in a variety of well numbers anddesigns. Most common are 96 and 384 well plates.

The well format will be determined by the end users needs, but it canhave numerous configurations and the wells do not necessarily need to beall of the same shape or size. For example, the wells of the presentinvention may have round, rectangular, teardrop, square, polygonal andother cross-sectional shapes or combinations of them. Virtually anyshape that is required for the product may be provided. Typically, ithas the wells arranged in uniformly spaced rows and columns for ease ofuse.

Ultrafiltration (UF) filters, which may be used in this process, can beformed from the group including but not limited to polysulphones,including polysulphone, polyethersulphone, polyphenylsulphones andpolyarylsulphones, polyvinylidene fluoride, and cellulose and itsderivatives, such as nitrocellulose and regenerated cellulose. Thesefilters typically include a support layer that is generally formed of ahighly porous structure. Typical materials for these support layersinclude various non-woven materials such as spun bounded polyethylene orpolypropylene, paper or glass or microporous materials formed of thesame or different polymer as the filter itself. Alternatively, thesupport may be an openly porous, asymmetric integral portion of theultrafiltration filter that may either be formed with or withoutmacrovoids. Such filters are well known in the art, and are commerciallyavailable from a variety of sources such as Millipore Corporation ofBedford, Mass.

Preferred UF filters include regenerated cellulose or polysulphonefilters such as YM™ or Biomax® filters available from MilliporeCorporation of Bedford, Mass.

Representative suitable microporous filters include nitrocellulose,cellulose acetate, regenerated cellulose, polysulphones includingpolyethersulphone and polyarylsulphones, polyvinylidene fluoride,polyolefins such as ultrahigh molecular weight polyethylene, low densitypolyethylene and polypropylene, nylon and other polyamides, PTFE,thermoplastic fluorinated polymers such as poly (TFE-co-PFAVE),polycarbonates or particle filled filters such as EMPORE® filtersavailable from 3M of Minneapolis, Minn. Such filters are well known inthe art and available from a variety of sources, such as DURAPORE®filters and EXPRESS® filters available from Millipore Corporation ofBedford, Mass.

The filter material may also be formed of glass fibers or mats, wovenplastics and non-woven plastics such as TYPAR® non-wovens available fromDuPont de Nemours of Wilmington. Del.

The filter may be in the form of an isotropic, track etched material(such as ISOPORE™ membranes), a cast membrane, preferably a microporousor ultrafiltration membrane such as DURAPORE® membranes, EXPRESS®membranes or EXPRESS® PLUS membranes available from MilliporeCorporation of Bedford, Mass., non-woven filter materials such as spunbonded polypropylene, polyethylene or polyester (Typar® or Tyvek®paper), PTFE resin membranes and the like.

A variety of adhesive bonding processes are envisioned and include lightcuring, air curing, hot melt adhesion, solvent adhesion and other suchmethods as are well known to one of ordinary skill in the art. Those ofordinary skill in the art would appreciate other means of adhering twolayers together.

The adhesive can be any one that is capable of bonding the filter to theplate bottom. Suitable adhesives include but are not limited to solventbased adhesives, crosslinking adhesives, such as room temperaturevulcanizable silicones, epoxies, including light curable epoxies such asUV light curable epoxies, hot melt adhesives and the like.

Preferably, a rapid curing adhesive such as a light curing,cyanoacrylate or thermally activated adhesives are preferred because theproduct can move continuously through a manufacturing process withoutthe requirements of batch processing. The light curing adhesives aremore preferred as the adhesive for attaching the filter to the plate.This is because this type of adhesive has been found to provide a liquidtight seal with a large variety of filters and plate materials and to doso in a continuous manufacturing process. The light curing adhesivessuch as 3201 and 3211 from Loctite Corporation works well. Other lightcuring adhesives are well known and readily available from companiessuch as Dymax of Torrington Conn., Masterbond of Hackensack, N.J.,Permabond of Engelwood, N.J. and others.

While light cured adhesives are preferred due to their ease of use,other adhesive systems such as two part epoxies and solvent basedadhesive systems can be used successfully in the invention especiallywhen the materials are found to be compatible.

When using adhesives it is required that the adhesive be suitable forbonding to both the plastic part and to the filter and not have anyadverse effect on the assay or filter performance.

It is preferred to use adhesives with relatively high viscosity(typically greater than 5000 cps, preferably greater than 7500 cps andmore preferably about 10,000 cps), so that the adhesive does not migrateto areas of the filter that otherwise would be used in the filtrationprocess. Any adhesive that migrates outside the seal area will reducethe effective filter area. One high viscosity adhesive is the Loctite3211 and it has been found suitable for use in this invention.

Alternatively, one may use lower viscosity adhesives in combination withthe use of masks to prevent the flow of adhesives to the area ofeffective filter. One may also form a series of troughs in the bottom ofthe plate to hold the adhesive and have the filter placed on top of thetroughs to contact the adhesive in the proper areas.

The method of forming the structure of the present invention is to useheat or a combination of heat and pressure to selectively collapseporous areas of the filter that lie over the solid portions of thebottom of the plate and to maintain the porous structure of the filterthat lies over the wells of the plate. The selection of heat or heat andpressure is determined by the porous structure material and the desiresof the designer. It is preferred with polymeric materials to use acombination of heat and pressure in forming the device.

In a typical method for making the present invention, one determineswhich process one will use and then forms a template in the desiredshape and pattern for the desired product. For example, one simplymeasures the bottom of the plate on to which the filter is to beattached and forms a template for that dimension.

The template is then placed against the filter after it has been gluedto the plate and a choice of heat or heat and pressure is applied to thefilter for a period of time sufficient to form the collapse of theporous material in the areas where the filter lies over a solid portionof the plate. Typically, the template is a flat, solid surface althoughit doesn't need to be. Alternatively, the template may contain a patternthat corresponds to the solid surface of the plate to which the filteris sealed. For example in 384 well plates, the wells are typicallysquare and a grid-like template where the grids of the templatecorrespond to the solid portions of the plate can be used.

When using heat, one should select a temperature which is sufficient tocause the pores in the selected areas to collapse but not to cause thepores is the others areas to collapse. This allows the areas that areheated treated to be rendered substantially non-porous, preferablynon-porous. The specific temperature is dependent upon the polymer used.However, the temperature should be from well before the structure beginsto deform to the melting point of the structure. Alternatively, one canuse a temperature from about 25° C. to about 500° C., preferably fromabout 25° C. to about 300° C. and more preferably from about 50° C. toabout 200° C. for a time sufficient to cause collapse of the porousstructure. The time can vary depending on the temperature used but canbe in the range of about 1 second to 60 minutes, preferably betweenabout 1 seconds and about 30 minutes and more preferably between about 2seconds and about 10 minutes.

The use of a laser may alternatively be made in performing the heatbonding step. Any laser that provides the necessary level of heat may beused. One such device is a Synrad CO2 laser. The power at which it isused depends upon the materials involved, the laser selected and thedesired depth of the laser penetration. For the Synrad CO2 laser, apower of about 10 watts is sufficient to provide the desired effect.

When using the combination of heat with pressure, one should usesufficient pressure to cause the collapse of the pores in the selectedarea without adversely affecting the pores in the other areas. Theamount of pressure used can vary depending on the amount of surface areato be collapsed, time, temperature and the strength of the plate, butone can typically use from about 10 psi to about 1,000 psi.

The template can be made of a material normally used in heatingapplications. Metals such as stainless steel or aluminum are preferredas they easily conduct heat. Various plastics such PTFE, polyethylene,especially ultrahigh molecular weight polyethylene (UPE), polypropyleneor epoxies can be used to make templates as well. Other materials suchas fiberglass or carbon composites can be used to make templates. Allthat is required is that the material have sufficient strength and heatconductivity to withstand the use. The template may also have anon-stick surface such as a PTFE coating in order to ensure easy removalof the formed structure from the template.

The following example shows the formation and use of one embodiment ofthe present invention.

EXAMPLE 1

5 series of 10 Millipore Multiscreen® polystyrene plates were obtained.Each series was sealed with a HVPP filter available from MilliporeCorporation of Bedford, Mass. to each series of plates in the followingmanner:

-   -   (1) Glued only (3201 light curable adhesive from Loctite        Corporation);    -   (2) Glued, followed by a laser cut (using a Synrad CO2 laser)        around the periphery of each well;    -   (3) Heat sealed only, using two separate heat seal steps at        375° F. for 4 seconds at 70 psi with the plate being turned 180°        between seals.    -   (4) Same as (3) followed by a laser cutting (using a Synrad CO2        laser) around the periphery of each well; and    -   (5) Glued as in (1) followed by the heat sealing process of (3).

The integrity of the wells on each plate in each of the series wastested by applying a vacuum at 20″ mercury to the underside of themembrane.

15 microliters (15 μl) of liquid (MILLI-Q® water) was added to each welland a vacuum was applied to draw the liquid through the filter into acollection well positioned below it. A product was deemed to be suitableif 12 or more μl of liquid was collected in the collection plate.

For Series 1 and 2 liquid collection was below 12 μl and liquid wasfound to migrate laterally in the filter between wells and to collect indead spots in the filter.

For Series 3 and 4, some wells collected 12 or more μl liquids, butlateral movement in the filter was still a problem.

For Series 5, which corresponds to the present invention, all wells wereintegral and passed at least 12 μl of liquid to the collection wells. Inaddition, no liquid was found to have moved laterally in the filter norwas any liquid found in the dead space of the filter (filter areabetween the wells).

EXAMPLE 2

Two 384 well plates, Multiscreen® polystyrene plates, available fromMillipore Corporation of Bedford, Mass., were obtained. Each was sealedwith a HVPP filter available from Millipore Corporation of Bedford,Mass. by a gluing procedure using 3201 light curable adhesive fromLoctite Corporation. The glue was applied to the ribs of the bottomsurface of the plates and then the membrane was applied over it. Handpressure was applied to obtain a good seal and the glue was cured withUV light.

One plate A was then heat sealed by a laser cut (using a Synrad CO2laser at an energy level of 10.4 watts) around the periphery of eachwell. The other plate B was left as is.

100 μl of a 0.1% (w/v) Ponceau S in 5% (w/v) acetic acid (Sigma Catalog# P7170) was added to one well of each plate. The liquid was allowed tostand at room temperature for fifteen minutes.

Any remaining liquid was then removed by pipette from the well and theplate was turned over and the bottom surface was observed andphotographed. FIG. 3A shows plate A according to the present invention.FIG. 3B shows plate B.

As can be clearly seen from FIGS. 3A and B, the plate according to theinvention (FIG. 3B) is only stained in the area of the one well thatcontained the solution. The well of B allowed fluid to spread laterallythrough the filter to adjoining wells (53 wells at least partiallycolored).

EXAMPLE 3

Two 345 well plates, each containing 96 active wells, Multiscreen®polystyrene plates, available from Millipore Corporation of Bedford,Mass., were obtained. FIG. 5 shows such an arrangement of the plate. Inthis design, only one well, 100, of the four adjacent wells 101, 102,103, is active, the others being covered by plastic to render themunusable. Each plate was sealed with a HVPP filter available fromMillipore Corporation of Bedford, Mass. by a gluing procedure using 3201light curable adhesive from Loctite Corporation. The glue was applied tothe ribs of the bottom surface of the plates and then the membrane wasapplied over it. Hand pressure was applied to obtain a good seal and theglue was cured with UV light.

One plate A was then heat sealed by a flat heater of a size and shape tofit the bottom surface of the plate in two separate heat seal steps at375° F. for 4 seconds at 70 psi with the plate being turned 180° betweenseals around the periphery of each well. The other plate B was left asis.

100 μl of a 0.1% (w/v) Ponceau S in 5% (w/v) acetic acid, (Sigma Catalog# P7170) was added to one well of each plate. The liquid was allowed tostand at room temperature for fifteen minutes.

Any remaining liquid was then removed by pipette from the well and theplate was turned over and the bottom surface was observed. FIG. 4A showsplate A according to the present invention. FIG. 4B shows plate B.

As can be clearly seen from FIGS. 4A and B, the plate according to theinvention (FIG. 4A) is only stained in the one well that contained thesolution. The well of 4B allowed fluid to spread laterally through thefilter to 87 (fully or at least partially) adjoining wells.

1. A method of forming a multiwell filtration plate comprising the stepsof: a) providing a multiwell plate containing a series of wells formedthrough the thickness of the plate, the plate having a top and a bottomsurface and the wells having an open top corresponding with the topsurface of the plate and an open bottom corresponding with the bottomsurface of the plate; b) applying glue to the bottom surface of theplate surrounding each well; c) providing a filter sheet of a size equalto or greater than that of the bottom surface of the plate and applyingthe filter sheet to the bottom of the plate; d) allowing the glue topenetrate the filter material; e) allowing the glue to cure; and f) thenheat bonding the filter material overlaying the bottom surface of theplate surrounding the wells.
 2. The process of claim 1 wherein the heatbonding is applied by a heated template corresponding in size and shapeto the bottom surface of the plate surrounding the wells, such that theheat bonding is formed in area beyond an outer periphery of each well.3. The process of claim 1 further comprising (g) attaching an underdrainto the bottom of the plate over the glued and heat sealed filtermaterial.
 4. A method of forming a multiwell filtration plate comprisingthe steps of: a) providing a multiwell plate containing a series ofwells formed through the thickness of the plate, the plate having a topand a bottom surface and the wells having an open top corresponding withthe top surface of the plate and an open bottom corresponding with thebottom surface of the plate; b) applying glue to the bottom surface ofthe plate surrounding each well; c) providing a filter sheet of a sizeequal to or greater than that of the bottom surface of the plate andapplying the filter sheet to the bottom of the plate; d) allowing theglue to penetrate the filter material; e) curing the glue; and f) thenheat bonding the filter material overlaying the bottom surface of theplate surrounding the wells so as to render the heat treated areassubstantially non-porous.
 5. The method of claim 4 wherein the heatbonding is by a heated platen corresponding in size and shape to thebottom surface of the plate surrounding the wells, such that the heatbonding is formed in area beyond an outer periphery of each well.
 6. Themethod of claim 4 wherein the heat treated areas are non-porous.
 7. Amethod of forming a multiwell filtration plate comprising the steps of:a. providing a multiwell plate containing a series of wells formedthrough the thickness of the plate, the plate having a top and a bottomsurface and the wells having an open top corresponding with the topsurface of the plate and an open bottom corresponding with the bottomsurface of the plate; b) applying glue selected from the groupconsisting of light curing adhesives, cyanoacrylate adhesives andthermally activated adhesives to the bottom surface of the platesurrounding each well; c) providing a filter sheet of a size equal to orgreater than that of the bottom surface of the plate and applying thefilter sheet to the bottom of the plate; d) allowing the glue topenetrate the filter material surrounding each well; e) allowing theglue to cure; and f) then heat bonding the filter material overlayingthe bottom surface of the plate surrounding the wells so as to renderthe heat treated areas non-porous.
 8. A method of forming a multiwellfiltration plate comprising the steps of: a. providing a multiwell platecontaining a series of wells formed through the thickness of the plate,the plate having a top and a bottom surface and the wells having an opentop corresponding with the top surface of the plate and an open bottomcorresponding with the bottom surface of the plate; b) applying glue tothe bottom surface of the plate surrounding each well; c) providing afilter sheet of a size equal to or greater than that of the bottomsurface of the plate and applying the filter sheet to the bottom of theplate; d) allowing the glue to penetrate the filter material; e)allowing the glue to cure; and f) then heat bonding the filter materialoverlaying the bottom surface of the plate surrounding the wells,wherein the heat bonding step of (f) is made by two sequential heatingsteps.
 9. A method of forming a multiwell filtration plate comprisingthe steps of: a) providing a multiwell plate containing a series ofwells formed through the thickness of the plate, the plate having a topand a bottom surface and the wells having an open top corresponding withthe top surface of the plate and an open bottom corresponding with thebottom surface of the plate; b) applying glue to the bottom surface ofthe plate surrounding each well; c) providing a filter sheet of a sizeequal to or greater than that of the bottom surface of the plate andapplying the filter sheet to the bottom of the plate; d) allowing theglue to penetrate the filter material; e) allowing the glue to cure; andf) then heat bonding the filter material overlaying the bottom surfaceof the plate surrounding the wells, wherein the heat bonding step of (f)is made by two sequential heating steps with the multiwell plate beingrotated 180 degrees between the sequential heating steps.
 10. A methodof forming a multiwell filtration plate comprising the steps of: a)providing a multiwell plate containing a series of wells formed throughthe thickness of the plate, the plate having a top and a bottom surfaceand the wells having an open top corresponding with the top surface ofthe plate and an open bottom corresponding with the bottom surface ofthe plate; b) applying glue to the bottom surface of the platesurrounding each well; c) providing a filter sheet of a size equal to orgreater than that of the bottom surface of the plate and applying thefilter sheet to the bottom of the plate; d) allowing the glue topenetrate the filter material; e) allowing the glue to cure; and f) thenheat bonding the filter material overlaying the bottom surface of theplate surrounding the wells so as to render the heat treated areassubstantially non-porous, wherein the heat bonding step of (f) is madeby two sequential heating steps.
 11. A method of forming a multiwellfiltration plate comprising the steps of: a. providing a multiwell platecontaining a series of wells formed through the thickness of the plate,the plate having a top and a bottom surface and the wells having an opentop corresponding with the top surface of the plate and an open bottomcorresponding with the bottom surface of the plate; b. applying glue tothe bottom surface of the plate surrounding each well; c. providing afilter sheet of a size equal to or greater than that of the bottomsurface of the plate and applying the filter sheet to the bottom of theplate; d. allowing the glue to penetrate the filter material; e.allowing the glue to cure; and f. then heat bonding the filter materialoverlaying the bottom surface of the plate surrounding the wells so asto render the heat treated areas substantially non-porous wherein theheat bonding step of (f) is made by two sequential heating steps withthe multiwell plate being rotated 180 degrees between the sequentialheating steps.
 12. A method of forming a multiwell filtration platecomprising the steps of: a) providing a multiwell plate containing aseries of wells formed through the thickness of the plate, the platehaving a top and a bottom surface and the wells having an open topcorresponding with the top surface of the plate and an open bottomcorresponding with the bottom surface of the plate; b) applying glueselected from the group consisting of light curing adhesives,cyanoacrylate adhesives and thermally activated adhesives to the bottomsurface of the plate surrounding each well; c) providing a filter sheetof a size equal to or greater than that of the bottom surface of theplate and applying the filter sheet to the bottom of the plate; d)allowing the glue to penetrate the filter material surrounding eachwell; e) allowing the glue to cure; and f) then heat bonding the filtermaterial overlaying the bottom surface of the plate surrounding thewells so as to render the heat treated areas non-porous, wherein theheat bonding step of (f) is made by two sequential heating steps.
 13. Amethod of forming a multiwell filtration plate comprising the steps of:a) providing a multiwell plate containing a series of wells formedthrough the thickness of the plate, the plate having a top and a bottomsurface and the wells having an open top corresponding with the topsurface of the plate and an open bottom corresponding with the bottomsurface of the plate; b) applying glue selected from the groupconsisting of light curing adhesives, cyanoacrylate adhesives andthermally activated adhesives to the bottom surface of the platesurrounding each well; c) providing a filter sheet of a size equal to orgreater than that of the bottom surface of the plate and applying thefilter sheet to the bottom of the plate; d) allowing the glue topenetrate the filter material surrounding each well; e) allowing theglue to cure; and f) then heat bonding the filter material overlayingthe bottom surface of the plate surrounding the wells so as to renderthe heat treated areas non-porous, wherein the heat bonding step of (f)is made by two sequential heating steps with the multiwell plate beingrotated 180 degrees between the sequential heating steps.